CN106164290A

CN106164290A - MIRNA ratio is used to determine lung cancer

Info

Publication number: CN106164290A
Application number: CN201480076735.0A
Authority: CN
Inventors: G.索兹; M.贝里; U.帕斯托里诺
Original assignee: Baio Milner Holdings Ltd
Current assignee: Baio Milner Holdings Ltd; BioMirna Holdings Ltd
Priority date: 2014-01-05
Filing date: 2014-12-31
Publication date: 2016-11-23
Also published as: JP2017502699A; EP3090059B1; JP2019122412A; US20170204467A1; CA2935789A1; WO2015101653A1; EP3090059A1; US20150191794A1; EP3425066A1; BR112016015595A2; AU2014375224A1; IL246580B; US20210301350A1; JP6706208B2; IL246580A0; US20190119756A1; ES2686631T3

Abstract

The present invention provides the method determining the existence situation of lung neoplasm in experimenter.The method determining the existence situation of aggressive lung neoplasm in experimenter is also provided.Additionally, it is provided that determine the method for the risk that lung neoplasm occurs in experimenter.The method determining the risk occurring aggressive lung neoplasm in experimenter is also provided.The method of the risk developing or having lung neoplasm is also provided in prediction experimenter.The method establishing lung cancer therapy option is additionally provided.

Description

MIRNA ratio is used to determine lung cancer

Cross reference to related applications

This application claims what the U.S. Provisional Application No. 61/923,758 submitted on January 5th, 2014 and on January 12nd, 2014 submitted to U.S. Provisional Application No. 61/926, the priority of 323 and rights and interests, the two application is all incorporated integrally into herein with it by quoting In.

Technical field

Present invention relates generally to diagnosing and determine the risk of lung cancer.More specifically, the application is directed to use with miRNA Express ratio and determine that lung neoplasm or the risk of aggressive lung neoplasm occur, and determine the existence feelings of lung neoplasm or aggressive lung neoplasm Condition.

Background of invention

Lung cancer is the main cause (Jemal etc., CA Cancer J Clin, 61:69-9,2011) of whole world cancer mortality. Current most of lung cancer is detected late, and at this moment treatment has limited efficacy and survival rate is low.Detect lung cancer in early days can show Write and reduce the death rate.

Europe randomization lung cancer screening test, utilizes and observes control group but limited size, rate of not being confirmed death up to now Reduce (Infante etc., Am J Respir Crit Care Med 180:445-453,2009;Saghir etc., Thorax 67:296-301, 2012;Pastorino etc., Eur J Cancer Prev 21:308-315,2012)；But, from The result of the national lung screening test (NSLT) that big NCI-initiates shows, with low dosage computerized tomography (LDCT) sieve Choosing has >=history in 30 Bao-year and self-stopping technology smoking since≤excessive risk of 15 years is individual, with annual chest radiography Art compares, and the death rate reduces by 20% (Aberle etc., N Engl J Med 365:395-409,2011).High false positive Rate, the cost (being estimated as $ 3,500,000 in the U.S.) screening the excessive risk individuality of high quantity and the potential hazard relevant with LDCT screening Remain consideration under clinical condition (Aberle etc., 2011;Goulart etc., J Natl Compr Canc Netw 10:267-275, 2012)。

Therefore, needs are existed to the other method of prediction, prognosis and the diagnosis improving lung cancer.The present invention meets described need Want.

Invention summary

The present invention is based in part on following discovery: assay method available circulation miRNA biomarker is with to overall " development or tool Have the risk of lung neoplasm " offer three-horizontal classification symbol is provided.

Therefore, in some embodiments, the method for the existence situation of lung neoplasm in determination experimenter is provided.Described method Including:

A () measures the expression ratio of miRNA pair in the biological sample of experimenter；

B () compares the average specific expressing ratio and multiple corresponding miRNA pair from multiple control samples from step (a) The cutoff (cut-off value) that rate measures；

If c the expression ratio of miRNA pair of () step (a) exceedes the cutoff of step (b), then described ratio is just distributed Scoring, if or the expression ratio of miRNA pair of step (a) is not less than the cutoff of step (b), then non-to the distribution of described ratio Just mark；

D other miRNA to repeating step (a) to (c), is assigned to expressing ratio by () until (1) at least 9 miRNA Just mark, or (2) are assigned less than 9 miRNA and just comment to expressing ratio after comparing the expression ratio of 27 miRNA pair Point,

Wherein said miRNA is to including 106a/140-5p, 106a/142-3p, 126/140-5p, 126/142-3p, 133a/ 142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、142-3p/21、142-3p/221、142- 3p/30b or 320/660, or its inverse ratio；With

E the existence situation of lung neoplasm is classified by () by following: if (i) at least 9 miRNA are just assigned to expressing ratio Scoring, then for the positive；Or (ii) is assigned if fewer than 9 miRNA just marks to expressing ratio, then for feminine gender.

According to another embodiment, determine that the method for the existence situation of aggressive lung neoplasm in experimenter includes:

B () compares the average specific expressing ratio and multiple corresponding miRNA pair from multiple control samples from step (a) The cutoff that rate measures；

(d) to other miRNA to repeating step (a) to (c), until (1) at least 14 miRNA is allocated to expressing ratio Just mark, or (2) are just assigned to expressing ratio less than 14 miRNA after compare the expression ratio of 28 miRNA pair Scoring,

Wherein said miRNA to include 106a/16, the 106/660th, the 16/17th, the 16/320th, the 17/660th, 197/30b, 197/30c, 320/451st, 320/486-5p or 320/660, or its inverse ratio；With

E the existence situation of aggressive lung neoplasm is classified by () by following: if (i) at least 14 miRNA are to expression ratio quilt It is assigned with and just marks, then for the positive；Or (ii) is assigned if fewer than 14 miRNA just marks to expressing ratio, then for the moon Property.

According to further embodiment, determine that the method for the risk occurring lung neoplasm in experimenter includes:

(d) to other miRNA to repeating step (a) to (c), until (1) at least 10 miRNA is allocated to expressing ratio Just mark, or (2) after comparing the expression ratio of 27 miRNA pair, be assigned to expressing ratio less than 10 miRNA Just mark,

Wherein said miRNA to including 133a/92a, 15b/21,15b/30b, 15b/30c, 16/197 or 28-3p/451, or its Inverse ratio；With

E () is by the following classification of risks that will appear from lung neoplasm: if (i) at least 10 miRNA are assigned to expressing ratio Just mark, then for the positive；Or (ii) is assigned if fewer than 10 miRNA expression ratios and just marks, then for feminine gender.

According to other embodiments, determine that the method for risk aggressive lung neoplasm occur includes:

Wherein said miRNA is to including 106a/142-3p, 126/142-3p, the 126/21st, 126/92a, 142-3p/17,142- 3p/197,142-3p/28-3p, 197/19b, 197/660 or 28-3p/660, or its inverse ratio；With

E () is by the following classification of risks that will appear from aggressive lung neoplasm: if (i) at least 14 miRNA are to expression ratio quilt It is assigned with and just marks, then for the positive；Or (ii) is assigned if fewer than 14 miRNA just marks to expressing ratio, then for the moon Property.

In other embodiments, it was predicted that the method for the risk developing in experimenter or having lung neoplasm includes:

A () determines the existence situation label of lung neoplasm mentioned above；

B () determines the existence situation label of aggressive lung neoplasm mentioned above；

C () determines the risk label of appearance lung neoplasm mentioned above；

D () determines the risk label of appearance aggressive lung neoplasm mentioned above；

If e none is classified as the positive in the label of () step (a)-(d), then experimenter is categorized as development or there is lung The low-risk of tumour；

If f the existence situation label of the lung neoplasm of () at least step (a) is the positive, or the wind of the appearance lung neoplasm of step (c) Danger label is the positive；And the appearance aggressive lung neoplasm of the existence situation label of the aggressive lung neoplasm of step (b) and step (d) Both risk labels be feminine gender, then experimenter is categorized as having development or there is the medium risk of lung neoplasm；With

If g the existence situation label of the aggressive lung neoplasm of () at least step (b) is positive or step (d) appearance invasion and attack Property lung neoplasm risk label be the positive, then experimenter is categorized as development or there is the excessive risk of lung neoplasm.

The method establishing treatment option to experimenter is additionally provided.Described method includes using the test of any of above method to be subject to Examination person and the result determination treatment option according to described method.Method can farther include to give treatment to need described treatment Experimenter.

Although present disclosure is described already in connection with its detailed description, but description above be intended to explanation and not Limiting the scope of the disclosure, it is defined by the scope of appended claims.Other side, advantage and modification with After claims in the range of.

Herein cited patent and scientific literature provide the available knowledge of those skilled in the art.Herein cited institute United States Patent (USP) and announcement or unpub U.S. Patent application is had to be combined by quoting.Herein cited all disclosed outside State's patents and patent applications is combined by quoting.Herein cited Genbank and the NCBI submission being indicated by searching number Combined by quoting.Herein cited all other disclosed reference, file, manuscript and scientific literature are by quoting In conjunction with.

Brief description

Fig. 1 is the sketch of the feature of the sample sets describing embodiment 1, embodiment 1: the lung detection of multicenter Italy (Multicentric Italian Lung Detection, MILD) studies 2005-2012.

Fig. 2 be show according to miRNA labeling accord with (MSC) in all experimenters from blood sample collect date The figure of three annual survival rates and table.

Detailed Description Of The Invention

According to an embodiment, the present invention utilizes multiple ratios of miRNA expression to determine that lung neoplasm occurs in (a) Risk, there is the risk of aggressive lung neoplasm in (b), the existence situation of (c) lung neoplasm, the existence feelings of (d) invasive tumor Condition, and when the risk of detection in combination step (a)-(d), determine the overall risk or develop with lung neoplasm.

The present invention can include the sample removing the haemolysis with detectable level, such as by including using miRNA haemolysis Classifier；Three-level (low, medium or high) " disease risks " classifier is used to replace two-level (low, high) classifier；Or make Replace storehouse (pool) with the comparison plasma sample from single experimenter.Can include three miRNA:miR-101, miR-145 and MiR-133a, its high variability in the verification step of mensuration before because in comparison storehouse is excluded.

According to the exemplary method of the present invention, " microRNA labeling symbol " (MSC) provides 87% to independent MSC Screening sensitiveness, and be 98% when with low dosage computerized tomography (LDCT) screening combination.Therefore, MSC can individually make With, or combine with other methods (such as LDCT) with cooperative mode, with by avoiding more wheels in the experimenter of vast scale LDCT and the diagnosis of unnecessary aggressive are followed up a case by regular visits to, and improve the validity that lung cancer is screened by LDCT.The MSC describing in detail in the application is independent Prognosis and diagnosis performance show, do not rely on tumor stage, in MSC measurement miRNA be not only the defeated of tumor burden Go out, but the pathogenetic indicant relevant with tumor invasiveness.

The computer side without bias of 4,950 ratios based on 100 different blood plasma miRNA of screening for the exploitation of MSC Method, to select optimal set (Boeri etc., the Proc of the miRNA ratio for the lung cancer detection and association with poor prognosis Natl Acad Sci USA 108:3713-3718, 2011).These miRNA ratios can reflect the different cellular components of tumour Regulation between the competition mechanism of the miRNA regulation of interior miRNA and microenvironment around.Do not restrainted by any particular theory Tiing up, stroma cell is activated by the lung microenvironment of inflammation, discharges in specific miRNA extremely circulation, and it can functionally participate in and tumour Convert the regulation of relevant target gene.

MSC utilizes the sane determination method of the miRNA label in blood plasma-source.MSC have malignant disease is existed situation, The diagnosis performance of the risk of following malignant tumour and the ability of the lung brief summary with major part optimum LDCT-detection for the differentiation lung cancer.This Literary composition details the available specific label of MSC.

" the existence situation of lung neoplasm " label

In some embodiments, the method for the existence situation of lung neoplasm in determination experimenter is provided.Described method includes:

(d) to other miRNA to repeating step (a) to (c), until (1) at least 9 miRNA is allocated to expressing ratio Just mark, or (2) are assigned less than 9 miRNA and just comment to expressing ratio after compare the expression ratio of 27 miRNA pair Point,

In some embodiments, the miRNA of this determination method to include ratio 106a/140-5p, 106a/142-3p, 126/140-5p、126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、 142-3p/21,142-3p/221,142-3p/30b and 320/660 or its inverse ratio more than the 2nd, more than the 3rd, more than the 4th, more than the 5th, surpass Cross the 6th, more than the 7th, more than the 8th, more than the 9th, more than the 10th, more than the 11st, more than 12 or each.

In other embodiments, miRNA is to can farther include ratio 106a/660,106a/92a, the 126/660th, 140-5p/197、140-5p/28-3p、142-3p/145、142-3p/197、142-3p/28-3p、17/660、17/92a、197/ 660th, 197/92a, 19b/660 or 28-3p/660, or its inverse ratio.

In other embodiment again, miRNA is to including ratio 106a/140-5p, 106a/142-3p, 126/140- 5p、126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、142-3p/ 21、142-3p/221、142-3p/30b、320/660、106a/660、106a/92a、126/660、140-5p/197、140-5p/ 28-3p、142-3p/145、142-3p/197、142-3p/28-3p、17/660、17/92a、197/660、197/92a、19b/ 660 and 28-3p/660 or its inverse ratio at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16th, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or Each.Skilled artisan will appreciate that, utilize the some but not all of this 27 ratios to provide precise Identification tumour to exist expection The determination method of situation.

In some embodiments, the present invention is provided to detect the multiple primer of any one of 27 miRNA pair or spy Pin is used for determining the purposes in the diagnostic reagent of the existence situation of lung neoplasm in experimenter in preparation, and wherein said detection includes:

In some embodiments, the plurality of primer or probe are used for detecting miRNA to expression ratio 106a/140- 5p、106a/142-3p、126/140-5p、126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、142- 3p/15b, 142-3p/17,142-3p/21,142-3p/221,142-3p/30b and 320/660 or its inverse ratio more than the 2nd, exceeding 3rd, more than the 4th, more than the 5th, more than the 6th, more than the 7th, more than the 8th, more than the 9th, more than the 10th, more than the 11st, more than 12 or each.

In other embodiments, the plurality of primer or probe are further used for detection miRNA to expression ratio 106a/ 660、106a/92a、126/660、140-5p/197、140-5p/28-3p、142-3p/145、142-3p/197、142-3p/28- 3p, the 17/660th, 17/92a, the 197/660th, 197/92a, 19b/660 or 28-3p/660 or its inverse ratio.

In other embodiment again, the plurality of primer or probe are used for detecting miRNA to expression ratio 106a/ 140-5p、106a/142-3p、126/140-5p、126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、 142-3p/15b、142-3p/17、142-3p/21、142-3p/221、142-3p/30b、320/660、106a/660、106a/ 92a、126/660、140-5p/197、140-5p/28-3p、142-3p/145、142-3p/197、142-3p/28-3p、17/ 660th, 17/92a, the 197/660th, 197/92a, 19b/660 and 28-3p/660 or its inverse ratio at least 9, at least 10, at least 11, extremely Few 12nd, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, extremely Few 23rd, at least 24, at least 25, at least 26 or each.Skilled artisan will appreciate that, utilize this 27 ratios some but non-entirely Portion is by the expected determination method providing precise Identification tumour to there is situation.

In some embodiments, at least one primer in the plurality of primer and probe or probe can selectively be tied At least one miRNA of in conjunction sample miRNA pair.In some embodiments, include can for the plurality of primer or probe MiRNA in selective binding sample is to expression ratio 106a/140-5p, 106a/142-3p, 126/140-5p, 126/142- 3p、133a/142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、142-3p/21、142-3p/ 221、142-3p/30b、320/660、106a/660、106a/92a、126/660、140-5p/197、140-5p/28-3p、142- 3p/145,142-3p/197,142-3p/28-3p, the 17/660th, 17/92a, the 197/660th, 197/92a, 19b/660 and 28-3p/ 660 at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, extremely Few 19th, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or at least one of each At least one primer of miRNA or probe.

In some embodiments, the present invention is provided to determine the kit of the existence situation of lung neoplasm in experimenter. Kit includes the multiple primer of any one or the probe in preparing diagnostic reagent for detection 27 miRNA pair, and utilizes The plurality of primer or probe determine the specification of the existence situation of lung neoplasm in experimenter.Described specification includes:

In some embodiments, the multiple primer providing in kit or probe can be used for detection miRNA to be compared to expressing Rate 106a/140-5p, 106a/142-3p, 126/140-5p, 126/142-3p, 133a/142-3p, 140-5p/17,142-3p/ 148a, 142-3p/15b, 142-3p/17,142-3p/21,142-3p/221,142-3p/30b and 320/660 or its inverse ratio More than the 2nd, more than the 3rd, more than the 4th, more than the 5th, more than the 6th, more than the 7th, more than the 8th, more than the 9th, more than the 10th, more than the 11st, more than 12 or each Individual.

In other embodiments, the multiple primer providing in kit or probe are further useful for detection miRNA pair Express ratio 106a/660,106a/92a, the 126/660th, 140-5p/197,140-5p/28-3p, 142-3p/145,142-3p/ 197th, 142-3p/28-3p, the 17/660th, 17/92a, the 197/660th, 197/92a, 19b/660 or 28-3p/660, or its inverse ratio.

In other embodiment again, the multiple primer providing in kit or probe can be used for detection miRNA to table Reach ratio 106a/140-5p, 106a/142-3p, 126/140-5p, 126/142-3p, 133a/142-3p, 140-5p/17, 142-3p/148a、142-3p/15b、142-3p/17、142-3p/21、142-3p/221、142-3p/30b、320/660、 106a/660、106a/92a、126/660、140-5p/197、140-5p/28-3p、142-3p/145、142-3p/197、142- 3p/28-3p, the 17/660th, 17/92a, the 197/660th, 197/92a, 19b/660 and 28-3p/660 or its inverse ratio at least 9, at least 10th, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21st, at least 22, at least 23, at least 24, at least 25, at least 26 or each.

In some embodiments, the multiple primer providing in kit and at least one primer or the probe of probe can At least one miRNA of in selective binding sample miRNA pair.In some embodiments, provide in kit is multiple Primer or probe include can miRNA in selective binding sample to express ratio 106a/140-5p, 106a/142-3p, 126/140-5p、126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、 142-3p/21、142-3p/221、142-3p/30b、320/660、106a/660、106a/92a、126/660、140-5p/197、 140-5p/28-3p、142-3p/145、142-3p/197、142-3p/28-3p、17/660、17/92a、197/660、197/ At least the 9 of 92a, 19b/660 and 28-3p/660, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16th, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or At least one primer of at least one miRNA of each or probe.

" the existence situation of aggressive lung neoplasm " label

In other embodiments, the method for the existence situation of aggressive lung neoplasm in determination experimenter is provided.Described method bag Include:

In some embodiments, the miRNA of this determination method to include ratio 106a/16, the 106/660th, the 16/17th, 16/ 320th, the 17/660th, 197/30b, 197/30c, the 320/451st, 320/486-5p and 320/660 or its inverse ratio more than the 2nd, more than the 3rd, More than the 4th, more than the 5th, more than the 6th, more than the 7th, more than the 8th, more than 9 or each.

In other embodiments, miRNA is to can farther include ratio 106a/451,106a/486-5p, the 126/451st, 126/486-5p、126/660、140-5p/197、16/197、17/451、17/486-5p、197/451、197/486-5p、197/ 660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p, or its inverse ratio.

In other embodiment again, miRNA to include ratio 106a/16, the 106/660th, the 16/17th, the 16/320th, 17/ 660、197/30b、197/30c、320/451、320/486-5p、320/660、106a/451、106a/486-5p、126/451、 126/486-5p、126/660、140-5p/197、16/197、17/451、17/486-5p、197/451、197/486-5p、197/ 660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p or its inverse ratio are at least 14th, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25th, at least 26, at least 27 or each.Skilled artisan will appreciate that, utilize some of this 28 ratios but not all by expection Precise Identification invasive tumor is provided to there is the determination method of situation.

In other embodiments, the present invention is provided to detect the multiple primer of any one of 28 miRNA pair or spy Pin is used for determining the purposes in the diagnostic reagent of the existence situation of aggressive lung neoplasm in experimenter, wherein said detection in preparation Including:

In some embodiments, multiple primers or probe can be used for detection miRNA to express ratio 106a/16,106/ 660th, the 16/17th, the 16/320th, the 17/660th, 197/30b, 197/30c, the 320/451st, 320/486-5p and 320/660 or its inverse ratio More than the 2nd, more than the 3rd, more than the 4th, more than the 5th, more than the 6th, more than the 7th, more than the 8th, more than 9 or each.

In other embodiments, multiple primers or probe can be further used for detection miRNA to expression ratio 106a/ 451、106a/486-5p、126/451、126/486-5p、126/660、140-5p/197、16/197、17/451、17/486- 5p, the 197/451st, 197/486-5p, the 197/660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p, or its inverse ratio.

In other embodiment again, multiple primers or probe can be used for detection miRNA to express ratio 106a/16, 106/660、16/17、16/320、17/660、197/30b、197/30c、320/451、320/486-5p、320/660、106a/ 451、106a/486-5p、126/451、126/486-5p、126/660、140-5p/197、16/197、17/451、17/486- 5p, the 197/451st, 197/486-5p, the 197/660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or At least the 14 of 28-3p/486-5p or its inverse ratio, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21st, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each.Skilled artisan will appreciate that, utilize this Some but not all determination methods that expected offer precise Identification invasive tumor is existed situation of 28 ratios.

In some embodiments, at least one primer in multiple primers and probe or probe can selective binding samples At least one miRNA of in product miRNA pair.In some embodiments, multiple primers or probe include selectively tying Close miRNA in sample to express ratio 106a/16, the 106/660th, the 16/17th, the 16/320th, the 17/660th, 197/30b, 197/30c, 320/451、320/486-5p、320/660、106a/451、106a/486-5p、126/451、126/486-5p、126/660、 140-5p/197、16/197、17/451、17/486-5p、197/451、197/486-5p、197/660、197/92a、19b/ 451st, 19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p at least 14, at least 15, at least 16, at least 17, At least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each At least one primer of at least one individual miRNA or probe.

In other embodiments, the present invention is provided to determine the examination of the existence situation of aggressive lung neoplasm in experimenter Agent box.Kit includes the multiple primer of any one or the probe in preparing diagnostic reagent for detection 28 miRNA pair, and The plurality of primer or probe is utilized to determine the specification of existence situation of aggressive lung neoplasm in experimenter.Described explanation school bag Include:

In other embodiment again, multiple primers or probe can be used for detection miRNA to express ratio 106a/16, 106/660、16/17、16/320、17/660、197/30b、197/30c、320/451、320/486-5p、320/660、106a/ 451、106a/486-5p、126/451、126/486-5p、126/660、140-5p/197、16/197、17/451、17/486- 5p, the 197/451st, 197/486-5p, the 197/660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or At least the 14 of 28-3p/486-5p or its inverse ratio, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21st, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each.

In some embodiments, kit provide multiple primer and probe at least one primer or probe can At least one miRNA of in selective binding sample miRNA pair.In some embodiments, provide in kit is multiple Primer or probe include can miRNA in selective binding sample to express ratio 106a/16, the 106/660th, the 16/17th, 16/ 320、17/660、197/30b、197/30c、320/451、320/486-5p、320/660、106a/451、106a/486-5p、 126/451、126/486-5p、126/660、140-5p/197、16/197、17/451、17/486-5p、197/451、197/ 486-5p, the 197/660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p are extremely Few 14th, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, extremely Lack at least one primer or the probe of the 25th, at least 26, at least 27 or at least one miRNA of each.

Together with above-mentioned two diagnosis tag (" the existence situation of lung neoplasm " and " the existence situation of aggressive lung neoplasm ") Rise, two prognosis labels label that " risk of lung neoplasm occurs " and " risk of aggressive lung neoplasm occur " label are provided.This A little prognosis labels can be used for determining after obtaining sample any time in section, such as 3 months, 6 months, 12 months, 18 months, 24 The risk of any time of outside individual month, 36 months or these time periods or centre.

The label that " risk of lung neoplasm occurs "

Therefore, in further embodiment, the method determining the risk that lung neoplasm occurs in experimenter is provided.Described method Including:

If c the expression ratio of miRNA pair of () step (a) exceedes the cutoff of step (b), then described ratio is just distributed Scoring；If or the expression ratio of miRNA pair of step (a) is not less than the cutoff of step (b), then non-to the distribution of described ratio Just mark；

(d) to other miRNA to repeating step (a) to (c), until (1) at least 10 miRNA is allocated to expressing ratio Just mark, or (2) are just assigned to expressing ratio less than 10 miRNA after compare the expression ratio of 27 miRNA pair Scoring,

In some embodiments, the miRNA of this determination method to include ratio 133a/92a, 15b/21,15b/30b, 15b/30c, 16/197 and 28-3p/451 or its inverse ratio more than the 2nd, more than the 3rd, more than the 4th, more than 5 or each.

In other embodiments, miRNA to can farther include ratio 101/140-3p, 106a/451,106a/660, 106a/92a、126/660、133a/451、133a/660、140-3p/660、142-3p/15b、15b/451、15b/660、17/ 451st, the 17/660th, 17/92a, 197/19b, the 197/451st, the 197/660th, 197/92a, 19b/660,28-3p/660 or 320/660, Or its inverse ratio.

In other embodiment again, miRNA to include ratio 133a/92a, 15b/21,15b/30b, 15b/30c, 16/197、28-3p/451、101/140-3p、106a/451、106a/660、106a/92a、126/660、133a/451、133a/ 660、140-3p/660、142-3p/15b、15b/451、15b/660、17/451、17/660、17/92a、197/19b、197/ 451st, the 197/660th, 197/92a, 19b/660,28-3p/660 320/660 or its inverse ratio at least 10, at least 11, at least 12, At least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, At least 24, at least 25, at least 26 or each.Skilled artisan will appreciate that, utilize the some but not all of this 27 ratios to incite somebody to action The determination method of the expected risk providing precise Identification that lung neoplasm occurs.

In some embodiments, the present invention is provided to detect the multiple primer of any one of 27 miRNA pair or spy Pin is used for determining the purposes in the diagnosis of the risk occurring lung neoplasm in experimenter or prognostic agent, wherein said detection in preparation Including:

In some embodiments, multiple primers or probe can be used for detection miRNA to expression ratio 133a/92a, 15b/ 21st, 15b/30b, 15b/30c, 16/197 and 28-3p/451 or its inverse ratio more than the 2nd, more than the 3rd, more than the 4th, more than 5 or each Individual.

In other embodiments, multiple primers or probe can be further used for detection miRNA to expression ratio 101/ 140-3p、106a/451、106a/660、106a/92a、126/660、133a/451、133a/660、140-3p/660、142- 3p/15b、15b/451、15b/660、17/451、17/660、17/92a、197/19b、197/451、197/660、197/92a、 19b/660,28-3p/660 or 320/660, or its inverse ratio.

In other embodiment again, multiple primers or probe can be used for detection miRNA to express ratio 133a/92a, 15b/21、15b/30b、15b/30c、16/197、28-3p/451、101/140-3p、106a/451、106a/660、106a/ 92a、126/660、133a/451、133a/660、140-3p/660、142-3p/15b、15b/451、15b/660、17/451、 17/660th, 17/92a, 197/19b, the 197/451st, the 197/660th, 197/92a, 19b/660,28-3p/660 320/660 or its At least the 10 of inverse ratio, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, At least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or each.Skilled artisan will appreciate that, profit By some but not all determination methods by the expected risk providing precise Identification that lung neoplasm occurs of this 27 ratios.

In some embodiments, at least one primer in multiple primers and probe or probe can selective binding samples At least one miRNA of in product miRNA pair.In some embodiments, multiple primers or probe include selectively tying Close miRNA in sample to express ratio 133a/92a, 15b/21,15b/30b, 15b/30c, the 16/197th, 28-3p/451, 101/140-3p、106a/451、106a/660、106a/92a、126/660、133a/451、133a/660、140-3p/660、 142-3p/15b、15b/451、15b/660、17/451、17/660、17/92a、197/19b、197/451、197/660、197/ 92a, 19b/660,28-3p/660 or 320/660 at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, extremely Few 16th, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 Or at least one primer of at least one miRNA of each or probe.

In other embodiments, the present invention is provided to determine the kit of the risk that lung neoplasm occurs in experimenter. Kit includes the multiple primer of any one or the probe in preparation diagnosis or prognostic agent for detection 27 miRNA pair, Specification with the risk utilizing the plurality of primer or probe to determine in experimenter to occur lung neoplasm.Described specification includes:

In other embodiment again, multiple primers or probe can be used for detection miRNA to express ratio 133a/92a, 15b/21、15b/30b、15b/30c、16/197、28-3p/451、101/140-3p、106a/451、106a/660、106a/ 92a、126/660、133a/451、133a/660、140-3p/660、142-3p/15b、15b/451、15b/660、17/451、 17/660th, 17/92a, 197/19b, the 197/451st, the 197/660th, 197/92a, 19b/660,28-3p/660 320/660 or its At least the 10 of inverse ratio, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, At least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or each.

In some embodiments, kit provide multiple primer and probe at least one primer or probe can At least one miRNA of in selective binding sample miRNA pair.In some embodiments, provide in kit is multiple Primer or probe include can miRNA in selective binding sample to express ratio 133a/92a, 15b/21,15b/30b, 15b/30c、16/197、28-3p/451、101/140-3p、106a/451、106a/660、106a/92a、126/660、133a/ 451、133a/660、140-3p/660、142-3p/15b、15b/451、15b/660、17/451、17/660、17/92a、197/ 19b, the 197/451st, the 197/660th, 197/92a, 19b/660,28-3p/660 or 320/660 at least 10, at least 11, at least 12, At least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, At least one primer of at least 24, at least 25, at least 26 or at least one miRNA of each or probe.

The label that " risk of aggressive lung neoplasm occurs "

The method that determine risk that aggressive lung neoplasm in experimenter occur is also provided herein.Described method includes:

In some embodiments, the miRNA of this determination method to include ratio 106a/142-3p, 126/142-3p, 126/ 21st, 126/92a, 142-3p/17,142-3p/197,142-3p/28-3p, 197/19b, 197/660 and 28-3p/660 or it is anti- Ratio more than the 2nd, more than the 3rd, more than the 4th, more than the 5th, more than the 6th, more than the 7th, more than the 8th, more than 9 or each.

In other embodiments, miRNA to can farther include ratio 106a/451, the 126/451st, the 145/197th, 17/ 451、197/21、197/30b、197/30c、197/451、197/92a、19b/451、21/221、21/28-3p、28-3p/30b、 28-3p/30c, 28-3p/451,28-3p/92a, 320/451 or 320/92a, or its inverse ratio.

In other embodiment again, miRNA is to including ratio 106a/142-3p, 126/142-3p, the 126/21st, 126/92a、142-3p/17、142-3p/197、142-3p/28-3p、197/19b、197/660、28-3p/660、106a/451、 126/451、145/197、17/451、197/21、197/30b、197/30c、197/451、197/92a、19b/451、21/221、 21/28-3p, 28-3p/30b, 28-3p/30c, 28-3p/451,28-3p/92a, 320/451 and 320/92a or its inverse ratio are extremely Few 14th, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, extremely Few 25th, at least 26, at least 27 or each.Skilled artisan will appreciate that, utilize the some but not all by advance of this 28 ratios There is the determination method of the risk of invasive tumor in phase offer precise Identification.

In some embodiments, the present invention is provided to detect the multiple primer of any one of 28 miRNA pair or spy Pin is used for determining the purposes in the diagnosis of the risk occurring aggressive lung neoplasm in experimenter or prognostic agent, Qi Zhongsuo in preparation State detection to include:

In some embodiments, multiple primers or probe can be used for detection miRNA to express ratio 106a/142-3p, 126/142-3p, the 126/21st, 126/92a, 142-3p/17,142-3p/197,142-3p/28-3p, 197/19b, 197/660 and 28-3p/660 or its inverse ratio more than the 2nd, more than the 3rd, more than the 4th, more than the 5th, more than the 6th, more than the 7th, more than the 8th, more than 9 or each.

In other embodiments, multiple primers or probe can be further used for detection miRNA to expression ratio 106a/ 451、126/451、145/197、17/451、197/21、197/30b、197/30c、197/451、197/92a、19b/451、21/ 221st, 21/28-3p, 28-3p/30b, 28-3p/30c, 28-3p/451,28-3p/92a, 320/451 or 320/92a, or it is anti- Ratio.

In other embodiment again, multiple primers or probe can be used for detection miRNA to expression ratio 106a/142- 3p、126/142-3p、126/21、126/92a、142-3p/17、142-3p/197、142-3p/28-3p、197/19b、197/ 660、28-3p/660、106a/451、126/451、145/197、17/451、197/21、197/30b、197/30c、197/451、 197/92a、19b/451、21/221、21/28-3p、28-3p/30b、28-3p/30c、28-3p/451、28-3p/92a、320/ 451 and 320/92a or its inverse ratio at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21st, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each.Skilled artisan will appreciate that, utilize this Some but not all determination methods by the expected risk providing precise Identification that aggressive lung neoplasm occurs of 28 ratios.

In other embodiments, the present invention is provided to determine the examination of the risk that aggressive lung neoplasm occurs in experimenter Agent box.Kit include preparation diagnosis or prognostic agent in for detection 28 miRNA pair the multiple primer of any one or Probe, and utilize the plurality of primer or probe to determine in experimenter to occur the specification of the risk of aggressive lung neoplasm.Described Specification includes:

In other embodiment again, multiple primers or probe can be used for detection miRNA to expression ratio 106a/142- 3p、126/142-3p、126/21、126/92a、142-3p/17、142-3p/197、142-3p/28-3p、197/19b、197/ 660、28-3p/660、106a/451、126/451、145/197、17/451、197/21、197/30b、197/30c、197/451、 197/92a、19b/451、21/221、21/28-3p、28-3p/30b、28-3p/30c、28-3p/451、28-3p/92a、320/ 451 and 320/92a or its inverse ratio at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21st, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each.

In some embodiments, kit provide multiple primer and probe at least one primer or probe can At least one miRNA of in selective binding sample miRNA pair.In some embodiments, provide in kit is multiple Primer or probe include can miRNA in selective binding sample to express ratio 106a/142-3p, 126/142-3p, 126/21、126/92a、142-3p/17、142-3p/197、142-3p/28-3p、197/19b、197/660、28-3p/660、 106a/451、126/451、145/197、17/451、197/21、197/30b、197/30c、197/451、197/92a、19b/ 451st, the 21/221st, 21/28-3p, 28-3p/30b, 28-3p/30c, 28-3p/451,28-3p/92a, 320/451 and 320/92a At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24th, at least one primer of at least 25, at least 26, at least 27 or at least one miRNA of each or probe.

The classifier that " develops or have the risk of lung neoplasm "

Above-mentioned four kinds of methods can combination in the method for prediction experimenter's development or the risk with lung neoplasm.The method includes:

A () determines the existence situation label of above-mentioned lung neoplasm；

B () determines the existence situation label of above-mentioned aggressive lung neoplasm；

C () determines the risk label of above-mentioned appearance lung neoplasm；

D () determines the risk label of above-mentioned appearance aggressive lung neoplasm；

If f the existence situation label of the lung neoplasm of () at least step (a) is the wind of the positive or the appearance lung neoplasm of step (c) Danger label is the positive；And the appearance aggressive lung neoplasm of the existence situation label of the aggressive lung neoplasm of step (b) and step (d) Both risk labels be feminine gender, then experimenter is categorized as having development or there is the medium risk of lung neoplasm；With

Above-mentioned four kinds of purposes can combination in the purposes of prediction experimenter's development or the risk with lung neoplasm.This purposes bag Include:

A () uses for detection 27 in the diagnostic reagent of the existence situation label prepared for determining above-mentioned lung neoplasm Multiple primer of any one of miRNA pair or probe；

B () uses for examining in the diagnostic reagent of the existence situation label prepared for determining above-mentioned aggressive lung neoplasm Survey the multiple primer of any one or the probe of 28 miRNA pair；

C () uses for examining in the diagnosis or prognostic agent of the risk label prepared for determining above-mentioned appearance lung neoplasm Survey the multiple primer of any one or the probe of 27 miRNA pair；

D () is used for determining use in the diagnosis of the risk label of above-mentioned appearance aggressive lung neoplasm or prognostic agent in preparation The multiple primer of any one or probe for detection 28 miRNA pair；

A. Definition

Terms used herein " lung neoplasm " can be optimum or malignant lung tumors.Lung neoplasm can have with one or more tuberculosis conditions Close, and can be in the form of such as lung brief summary or lung block.

Terms used herein " tuberculosis condition " refers to the change of the disease with regard to lung, event or health status, including for example Lung cancer and the various non-cancer patient's condition.The example of non-cancer tuberculosis condition includes chronic obstructive pulmonary disease (COPD), carcinoid lung tumor or cell Block (such as hamartoma, fibroma, neurofibroma), granuloma, sarcoidosis and by bacterium (such as tuberculosis) or fungi is (for example Histoplasmosis) infection that causes of pathogen.In certain embodiments, tuberculosis condition can be with the appearance of radiography lung brief summary Relevant.

Said method is not straitly limited to diagnosis and/or predicts any concrete lung cancer." lung cancer " used herein preferably Refer to the cancer of lung, but any disease or other illnesss of the respiratory system of people or other mammals can be included.Respiratory system swells Knurl illness includes such as small cell carcinoma or ED-SCLC (SCLC), non-small cell carcinoma or non-small cell lung cancer (NSCLC), squamous Cell cancer (SCC), gland cancer, bronchovesicular cancer (BAC), mixed type lung cancer, MPM, undifferentiated large cell carcinoma, Carcinoma gigantocellulare, synchronization knurl, maxicell neuroendocrine carcinoma, gland carcinoma squamosum, undifferentiated carcinoma；And small cell carcinoma, including oat cell Cancer, mixed type cellule/large cell carcinoma (LC), large cell carcinoma and combined small cell carcinoma；And adenoid cystic carcinoma, hamartoma, Mucoepidermoid tumor, typical carcinoid lung neoplasm, atypia carcinoid lung neoplasm, periphery carcinoid lung neoplasm, maincenter carcinoid lung neoplasm, chest Intermembranous rind gall, undifferentiated lung cancer and the cancer originating from outside lung are for example transferred to the secondary cancer of lung from the other parts of health Or currently known or that be later discovered that any other lung cancer.Lung cancer can be any stage or grade.

Terms used herein " from the biological sample of experimenter " refers to the sample from any experimenter, including smoking Person, non-smoker, from Ex smoker, cancer patient's (cancer return after for example for diagnosis and/or monitoring treatment), the past Cancer patient etc..In particular embodiments, biological sample derives from smoking individual, its gather sample when if carried out one-tenth Then there is not lung neoplasm as diagnostic method；Particularly when carry out CT scan then there is not size more than the brief summary of 5 mm Smoking individual.

Terms used herein " biological sample " can be to provide any group of the accurate measurement of the miRNA overview of experimenter Knit.In some embodiments, biological sample is biofluid.These embodiments are not straitly limited to any specific body Liquid, because miRNA is present in essentially all of body fluid (Weber etc., The microRNA spectrum in 12 body fluids, Clin Chem 56:1733-1741, 2010;De Guire etc., Clin Biochem 46:846- 860, 2013；Also Rodriguez-Dorantes etc., Meth Mol Biol 1165:81-87,2014 are seen).Useful The example of body fluid is peripheral blood, serum, blood plasma, ascites, urine, phlegm, saliva, BAL fluid, capsule liquid, liquor pleurae, abdomen Film liquid, lymph, purulence, the irrigating solution from hole room, broncho-pulmonary aspirate and bone marrow.Technical staff can be without excessively in fact Test and determine whether any specific body fluid can be used for any application-specific.In some embodiments, body fluid is blood plasma or serum. In some embodiments, biological sample is tissue sample.

Singulative used herein " one ", " one " and " described " are intended to also include plural form, unless civilization up and down Really additionally instruction.In addition, the use of "or" is intended to include "and/or", unless context clearly additionally indicates.

" individual " used herein, " experimenter ", " patient " or " experimenter in need " is to have development tumour or invade The individuality of the risk of attacking property tumour, or can have the individuality that or can suffer from or have after diagnosing tumour or invasive tumor.These Term is used interchangeably.Preferably, individuality is mammal.Mammal can be for example any mammal, such as people, spirit Long class animal, bird, mouse, rat, poultry, dog, cat, milk cow, horse, goat, camel, sheep or pig.Preferably, mammal is People.

" primer " or " probe " in multiple primer used herein or probe is nucleic acid molecules.Primer or probe can be DNA, RNA or cDNA.That primer or probe can be naturally-occurring or synthesis.Primer or probe can be at least 5, at least 10th, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100 nucleic acid are long.Preferably, in multiple primers and probe At least one primer or probe can at least one miRNA of in selective binding sample miRNA couple.Preferably, multiple Primer or probe comprise can in selective binding sample all miRNA at least one miRNA expressing ratio at least one Individual primer or probe.Term " selective binding " means that primer or probe have at least to miRNA pair combining in sample The high-affinity of individual miRNA, or compare at least one miRNA's combining miRNA couple with any other miRNA in sample More high-affinity.Term " primer " means that nucleic acid molecules can act as the starting point of DNA synthesis.I other words, archaeal dna polymerase can be from drawing 3 ' ends of thing start to replicate.

Using any of above method, terms used herein " the expression ratio of miRNA pair " is the expression of two specific miRNA The ratio of level.Should be understood that the ratio of any miRNA pair specified in any method as herein described can be as the ratio specified Inverse ratio calculate.As non-limiting examples, when the miRNA of concrete grammar to being " 106a/140-5p, 106a/142-3p, 126/140-5p、126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、 During 142-3p/21,142-3p/221,142-3p/30b and 320/660, or its inverse ratio ", these any 1 of miRNA pair, 2 Individual, 3,4,5,6,7,8,9,10,11, the expression ratio of 12 or 13 can calculate as inverse ratio, Wherein cutoff is adjusted accordingly.

Terms used herein " cutoff " can be negative or positive number.For some ratios, the table of " exceeding cutoff " Reaching ratio is quantitatively less than the value of cutoff.For other ratios, the expression ratio of " exceeding cutoff " is quantitatively Value more than cutoff.See for example table 9, wherein " > " represent that the number representative being quantitatively more than cutoff exceedes cutoff Ratio, and " < " represents that the quantitatively number representative less than cutoff exceedes the ratio of cutoff.

Under this definition " exceeding cutoff ", (i) exceedes in table 9 listed for the value-4.50 of ratio 133a/92a -4.18 cutoff, (ii) exceedes the cutoff of in table 9 listed 3.33, (iii) for the value 3.50 of ratio 17/451 The value-1.80 of ratio 142-3p/17 is exceeded to the cutoff of in table 9 listed-1.95, and (iv) is for ratio 16/197 Value 4.50 exceed in table 9 listed 5.00 cutoff.

In any method as herein described, the cutoff of the expression ratio of any specific miRNA pair is from multiple control samples The average ratio of multiple corresponding miRNA pair of product measures.Terms used herein " multiple control sample " may be from any really Fixed colony, the such as health volunteer without any cancer history, the health volunteer without lung cancer history, there is the tested of lung cancer The experimenter of person, never smoking, once smoking but the experimenter etc. of no longer smoking.In preferred embodiments, multiple comparisons Sample is from health volunteer, and cutoff is the average ratio from multiple corresponding miRNA pair of multiple control samples.? In some of these embodiments, terms used herein " mean value " can be average or median.In other embodiments In, cutoff is any smaller or greater ratio of one standard deviation of mean value +/-or standard deviation, for example +/-mark Quasi-deviation, +/-standard deviation, 1.5 standard deviations of +/-, 2 standard deviations of +/-or standard deviation any bigger, Less or in middle ratio.

For any number of control sample from any control population, cutoff can be not necessarily to by using known method Excessively experiment and determine, such as by using the training set (training set) described in embodiment.Establish cutoff to obtain Sensitiveness (SE), specific (SP), positive predictive value (PPV) and the negative predictive value (NPV) that must need.

For evaluating these diagnostic methods (existence situation of tumour；The existence situation of invasive tumor) and method of prognosis (go out The risk of existing tumour；The risk of invasive tumor occurs), the result of the test having analysed in clinical settings is dissolved in classification.Method Diagnosis or prognosis values can be defined by its SE, SP, PPV and NPV.Any test method will produce true positives (TP), false negative (FN), false positive (FP) and true negative (TN)." sensitiveness " of test be with the presence of disease or have a response there is positive test The percentage of all patients, or (TP/TP+FN) × 100%." specifically " of test is to have the moon without disease or without respond Property test the percentage of all patients, or (TN/FP+TN) × 100%." predicted value " or " PV " of test is that value is (positive or cloudy Property) it is the tolerance (%) of the number of times of actual value, it is i.e. that the percentage of all positive tests of true positives is positive predictive value (PV+) Or (TP/TP+FP) x100%." negative predictive value " (PV) be the patient with negative test being not responding to percentage or (TN/FN+TN)×100%.Another tolerance, " degree of accuracy " or " validity " of test, is that test is just given compared with test sum The percentage of the number of times of true answer, or (TP+TN/TP+TN+FP+FN) × 100%." error rate " from predicted have response and without sound Those patients and the predicted nothing answered respond and have those patients of response to calculate, or (FP+FN/TP+TN+FP+FN) × 100%. Overall test " specifically " is the tolerance of the degree of accuracy of sensitiveness, and the specific of test may not with the overall of disease in colony Property change and change, and predicted value can change.The presence or absence of or clinical of disease in given patient is rung by PV with doctor The presence or absence of clinical evaluation answered and change.

For any given test, TP, FN, FP and TN can be determined and adjusted by technical staff and without excessive experiment, lead to Cross and for example adjust cutoff, adjust the statistical significance (such as P value) for making prognosis or diagnositc decision；Adjustment Tests program Accuracy.

B. Method

The expression of miRNA can be measured by any method known in the art.In some embodiments, anti-by using The cDNA copy that transcriptase-polymerase chain reaction (RT-PCR) prepares each miRNA of each miRNA centering measures expression. In each embodiment, express ratio and use real-time PCR to measure further.In the specific embodiment of these methods, Expressing ratio uses TaqMan probe to measure further.But, described method is not limited to these embodiments, and can use now Any suitable method enforcement for determining miRNA expression that is known or that be later discovered that.

In some embodiments of the inventive method, before measurement, expand miRNA.In other embodiments, expanding The level of these nucleic acid is measured during increasing process.In other method again, not amplification of nucleic acid before measuring.

There is many methods and be used for expanding miRNA nucleotide sequence, for example ripe miRNA, precursor miRNA and primary miRNA. Suitable nucleic acid polymerization and amplification technique include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q- PCR)), based on the amplification (NASBA) of nucleotide sequence, ligase chain reaction, multichannel can linking probe amplification, invader technology (Third Wave), rolling circle amplification, in-vitro transcription (IVT), strand displacement amplification, the amplification (TMA) of transcriptive intermediate, RNA (Eberwine) amplification and other methods well known by persons skilled in the art.In certain embodiments, a kind of expansion is used more than Increasing method, such as reverse transcription, then real-time quantitative PCR (qRT-PCR).In PCR and q-PCR method, for example, to each target sequence Row use one group of primer.In certain embodiments, the length of primer depends on many factors, including but not limited to primer, target The hybridization temperature needing between nucleotide sequence and the complexity of different target nucleic acid sequences to be amplified.In certain embodiments, It is long that primer is about 15 to about 35 nucleotides.In other embodiments, the 15th, primer is equal to or less than the 20th, the 25th, 30 or 35 core Thuja acid is long.In another embodiment, primer is that at least 35 nucleotides are long.

Further, forward primer can comprise at least one sequence being annealed to miRNA, and alternatively can comprise 5 ' other incomplementarity districts.On the other hand, reverse primer can be designed to be annealed to the complement of the miRNA of reverse transcription.Instead MiRNA sequence can not relied on to primer, and multiple miRNA biomarker can use identical reverse primer to expand.Or, instead Can be special to miRNA biomarker to primer.

QRT-PCR reaction can by including reverse transcriptase and the heat-stable DNA polymerase based on DNA, further with Reverse transcription reaction combines.When using two kinds of polymerases, " thermal starting " method can be used to make determination method maximizing performance, and (U.S. is special Profit number 5,411,876 and 5,985,619).For example, the component of reverse transcriptase reaction and PCR reaction can use one or more heat Activiation method or chemical modification isolation, with improve polymerization effect (U.S. Patent number 5,550, the 044th, 5,413,924 and 6,403, 341)。

In certain embodiments, the probe of label, dyestuff or mark and/or primer are for that detect amplification or do not expand MiRNA.It will be recognized that according to the abundance of the sensitiveness of detection method and target, any detection method is suitable 's.Sensitiveness according to detection method and the abundance of target, can need or not need amplification before testing.Art technology Personnel will be appreciated that miRNA amplification is preferred detection method.

Probe or primer can include the base of Watson-Crick base or modification.The base modified includes but is not limited to AEGIS base (from Eragen Biosciences), it has been described in such as U.S. Patent number 5,432,272,5,965,364 With 6,001,983.In some aspects, base connects bonded by natural phosphodiester bond or different chemistry.Different changes Company's key includes but is not limited to peptide bond or lock nucleic acid (LNA) Lian Jian, and it is described in such as U.S. Patent number 7,060,809.

Further, present in amplified reaction, oligonucleotide probe or primer are suitable for monitoring and produce in time The amount of amplified production.In some aspects, there is the probe of different strand contrast double-strand proterties for detecting nucleic acid.Probe bag Include but be not limited to 5 '-exonuclease enzymatic determination (such as TaqMan) probe (seeing U.S. Patent number 5,538,848), stem-ring Molecular beacon (see for example U.S. Patent number 6,103,476 and 5,925,517), acaulescence or Linear Beacon (see for example WO 9921881st, U.S. Patent number 6,485,901 and 6,649,349), peptide nucleic acid (PNA) molecular beacon (see for example United States Patent (USP) Numbers 6,355,421 and 6,593,091), linear PNA beacon (see for example U.S. Patent number 6,329,144), non-FRET probe (see for example U.S. Patent number 6,150,097), Sunrise/AmplifluorB probe (see for example U.S. Patent number 6,548,250), stem-ring and duplex Scorpion probe (see for example U.S. Patent number 6,589,743), bulge loop probe (see for example U.S. Patent number 6,590,091), false knot probe (see for example U.S. Patent number 6,548,250), cyclicons (see for example U.S. Patent number 6,383,752), MGB Eclipse probe (Epoch Biosciences), hairpin probe (see for example U.S. Patent number 6,596,490), PNA illuminate probe, anti-primer quenching probe (Li etc., Clin. Chem. 53:624-633 (2006)), self-assembly nanoparticle probes and the ferrocene being described in such as U.S. Patent number 6,485,901 The probe modified.

In certain embodiments, one or more of amplified reaction primer includes label.Other embodiment party again In case, different probes or primer include diacritic detectable label each other.In some embodiments, nucleic acid, for example, visit Pin or primer, can use two or more diacritic labels mark.

In some embodiments, the concentration of miRNA uses microarray or the measurement of another carrier.

" microarray " is linear two-dimentional or three-dimensional (and solid phase) array of discrete regions, each has the table at solid carrier The determination region being formed on face, described carrier is such as, but not limited to glass, plastics or synthesis film.Discrete regions on microarray close The sum determination by the fixing polynucleotides to be detected on the surface of single solid phase carrier for the degree, for example, at least about 50/ cm², at least about 100/cm²Or at least about 500/cm², until about 1,000/cm²Or it is higher.Array can comprise altogether less than about 500th, about the 1000th, about the 1500th, about the 2000th, about the 2500th, about 3000 or more fixing polynucleotides.The micro-battle array of DNA used herein Arranging the array of the oligonucleotides being placed on chip or other surfaces or polynucleotide probes, it is used for being hybridized to from sample amplification Or the polynucleotides of clone.Because the position of each concrete probe groups is known on array, the identity of sample polynucleotide can Determine with the combination of the ad-hoc location on microarray according to them.

As the alternative using microarray, the array on carrier with any size can be used for implementing in the disclosure Hold, including the arrangement of one or more positions of two dimension or three-dimensional arrangement, to detect the expression of miRNA.

In some respects, label is connected to one or more probe, and label has one or more following character: I () provides detectable signal；(ii) interact with the second label to change the detectable signal that the second label provides, for example FRET (FRET)；(iii) stable hybridization, such as duplex is formed；(iv) provide combine compound or The member of affine group, such as affinity, antibody-antigene, ion complex, haptens-part (such as biotin-avidin). At other aspect again, the use of label can use any one in multiple known technology, uses known label, Lian Jian, company Connect group, reagent, reaction condition and analysis and purification process realizes.

MiRNA can be detected by direct or indirect method.In direct detection method, one or more miRNA lead to Cross the detectable label detection being connected with nucleic acid molecules.In such method, miRNA can enter rower with probe before being combined Note.Therefore, combination is detected by screening the miRNA of the mark being combined with probe.Pearl optionally and in reaction volume for the probe is even Connect.

In certain embodiments, nucleic acid is by the probe with mark directly in conjunction with detecting, and probe is detected subsequently. In one embodiment of the invention, nucleic acid, the miRNA for example expanding, use with probe conjugate to capture the nucleic acid of needs FIexMAP Microspheres (Luminex) detection.Certain methods can include the many nucleosides for example modified by fluorescence labels Acid probe detection or branched DNA (bDNA) detection.

In other embodiments, nucleic acid is detected by Indirect Detecting Method.For example, biotinylated probe can resist with strepto- The dye combinations of biotin-put together is to detect the nucleic acid of combination.Streptavidin molecule combines the biology on the miRNA of amplification Element label, and the miRNA combining detected by detecting the dye molecule that is connected with streptavidin molecule.An enforcement In scheme, the dye molecule of streptavidin-put together includes Phycolink streptavidin R-PE (PROzyme).Other dye molecules puted together are well known by persons skilled in the art.

Label includes but is not limited to: light is launched, light scattering and light-absorbing compound, its produce or quencher can detect fluorescence, Chemiluminescence or bioluminescence signal (see for example Kricka, L., Nonisotopic DNA Probe Techniques, Academic Press, San Diego (1992) and Garman A., Non-Radioactive Labeling, Academic Press (1997)).Include but is not limited to fluorescein as the fluorescent reporter molecule dyestuff of label (to see for example U.S. Patent number the 5,188,934th, 6,008,379 and 6,020,481), rhodamine (see for example U.S. Patent number the 5,366,860th, 5,847,162nd, the 5,936,087th, 6,051,719 and 6,191,278), phenonaphthazine (see for example U.S. Patent number 6,140, 500)；Energy transfer fluorescent dye, including donor and acceptor are to (see for example U.S. Patent number 5,863,727；5,800,996； With 5,945,526), and cyanine (see for example WO 9745539), Liz amine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5、Cy7、FluorX (Amersham)、Alexa 350、Alexa 430、AMCA、BODIPY 630/650、BODIPY 650/665、BODIPY-FL、BODIPY-R6G、BODIPY-TMR、BODIPY-TRX、Cascade Blue、Cy3、Cy5、6- FAM, fluorescein isothiocynate, HEX, 6-JOE, Oregon Green the 488th, Oregon Green the 500th, Oregon Green 514、Pacific Blue、REG、Rhodamine Green、Rhodamine Red、Renographin、ROX、SYPRO、 TAMRA, tetramethylrhodamin and/or Texas Red, and any other fluorescing fractions of detectable signal can be produced.Glimmering The example of fluorescein dye includes but is not limited to 6-Fluoresceincarboxylic acid；2 ', 4 ', 1,4 ,-tetrachlorofluorescein；With 2 ', 4 ', 5 ', 7 ', 1, 4-chlordene fluorescein.In some aspects, fluorescence labels selected from SYBR-Green, 6-Fluoresceincarboxylic acid (" FAM "), TET, ROX, VICTM and JOE.For example, in certain embodiments, label is can to launch the light of the analysable wavelength of different spectrum not Same fluorogen (such as 4-different colours fluorogen)；The probe of some such mark is to it known in the art, and be described in Literary composition and U.S. Patent number 6,140,054.Use in some embodiments and include reporter fluorescence group and quenching molecules fluorescence The fluorescence probe of the double labeling of group.It should be appreciated that selection has the fluorogen pair of different emission spectrum, in order to they can be easy It is distinguished.

It yet still another aspect, label is hybridization-stabilizing moiety, it is used for strengthening, stablizing or affect the hybridization of duplex, for example Intercalator and intercalative dye (including but not limited to ethidium bromide and SYBR-Green), minor groove binding and crosslinking functionality (ginseng See the editors " DNA and RNA Structure " such as such as Blackburn, be loaded in Nucleic Acids in Chemistry and Biology (1996))。

In other side, the method depending on hybridization and/or connection with quantitative miRNA can be used, including oligonucleotides is even Connect (OLA) method and allow to be hybridized to the method that the probe distinguished of target nucleic acid sequence separates with uncombined probe.As an example, The HARP-sample probe being disclosed in US publication 2006/0078894 can be used for measuring the quantity of miRNA.

In the other embodiments of described method, probe coupled reaction can be used for quantitative miRNA.Depend at multi-wad join In probe amplification (MLPA) technology relying (Schouten etc., Nucleic Acids Research 30:e57 (2002)), The probe of direct hybridization located adjacent one another on target nucleic acid is to being only connected to each other in the presence of target nucleic acid.In some respects, MLPA probe has side PCR primer binding site even.MLPA probe can only expand in the case that they have connected, therefore Allow detection and quantitative miRNA biomarker.

C. The detection of haemolysis

Red blood cell and hematoblastic haemolysis release miRNA, its may interfere with said method (Kirschner etc., PLoS One 6: e24145, 2011;Pritchard etc., Cancer Prev Res (Phila) 5:492-497,2012).Therefore, upper State in some embodiments of method, measure the haemolysis in biological sample, and if measure haemolysis, then sample does not carry out described side Method.

Haemolysis can be measured by any method known in the art in Samples subjects.In some embodiments, molten Blood is through spectrophotometry measurement, such as by measuring the absorbance (414nm, 541nm, 576nm) of different wave length to reflect In random sample product free hemoglobin existence situation and amount (Kirschner etc., 2011).

In other embodiments, by analyzing the miRNA that the haemolysis of increment regulation in the sample that haemolysis occurs is related to Expression measure haemolysis.In some of these embodiments, use miRNA miR-451, miR-486-5p, miR- 16th, any one in miR-92a or miR-140-3p, compared with the sample that haemolysis does not occurs, it is in the sample that haemolysis occurs Notable increment regulation.In the various aspects of these embodiments, the related miRNA of haemolysis be miR-451, miR-486-5p, MiR-16 and miR-92a.

In further embodiment, measure multiple correction miRNA of non-increment regulation in the sample that haemolysis occurs Expression with for normalizing the expression of miRNA related for haemolysis.These corrections miRNA can be related to haemolysis MiRNA ratio use with will measure the latter miRNA expression normalization.In some of these embodiments, Correction miRNA includes miR-126, miR-15b, miR-221 and miR-30b.

" haemolysis " label

In some embodiments, haemolysis is further by following mensuration:

A () determines in sample by each of miR-451, miR-486-5p, miR-16 and miR-92a with miR-126, miR- Expression ratio (the i.e. miR-of each of be composed of 16 miRNA pair of each of 15b, miR-221 and miR-30b 451/miR-126、miR-451/miR-15b、miR-451/miR-221、miR-451/miR-30b、miR-486-5p/miR- 126、miR-486-5p/miR-15b、miR-486-5p/miR-221、miR-486-5p/miR-30b、miR-16/miR-126、 miR-16/miR-15b、miR-16/miR-221、miR-16/miR-30b、miR-92a/miR-126、miR-92a/miR-15b、 MiR-92a/miR-221 and miR-92a/miR-30b or its inverse ratio)；

B () compares from multiple corresponding with to from multiple control samples of each of 16 of step (a) expression ratios Each of the average ratio of miRNA pair expresses the cutoff that ratio measures；

C () is for each of 16 miRNA pair, if the expression ratio of step (a) exceedes the cutoff of step (b), then right The distribution of described ratio is just marked, if or the expression ratio of step (a) is not less than the cutoff of step (b), then to described ratio Distribution anon-normal scoring；With

D sample is classified by () by following: if in (i) 16 ratios 8 or more exceed cutoff, then have molten Blood；Or (ii) is if exceeding cutoff less than 8 in 16 ratios, then do not have haemolysis.

With regard to above-mentioned diagnosis and prognostic assays, the cutoff of this Haemolytic Assay depends on control population, for example its In multiple blood serum samples that haemolysis do not occurs cutoff of being used as control sample will differ from the serum of plurality of generation haemolysis Sample is used as the cutoff of control sample.See embodiment.In addition, cutoff adjustable, for example with realize required SE, SP, PPV and NPV value.The blood serum sample that haemolysis does not occurs wherein is used as in some embodiments of control sample, and cutoff will be Average (average or median) ratio of multiple control samples.Optionally, this cutoff has the standard deviation of +/-ratio, with Explain for measuring the variable difference between the sample of cutoff.

In each embodiment, any Haemolytic Assay of above-mentioned use miRNA expression is also surveyed with another haemolysis Determine method combination, such as Kirschner etc., the spectrophotometry determination method described in 2011.

D. Diagnosis combination

As be shown in the examples, combine MSC label as herein described and allow inspection with low dosage computerized tomography (LDCT) Survey other cancers that can not be detected by LDCT, improve screening sensitiveness, from 87% and to independent LDCT 84% to independent MSC Improve to 98% during LDCT and MSC combination.With LDCT outside the combination of cancer screening test method when, above-mentioned diagnosis and pre- Rear method is by the similar improvement of expected display diagnostic sensitivity.

The health care cost of LDCT screening and related follow-up procedure is significant.Nearest consideration is extensively adopted in the U.S. Affecting model with the budget of LDCT to show, LDCT screening will avoid the up to 8100 too early lung cancer deaths of case, screening rate 75%, it is to avoid One case lung cancer death needs extra screening cost (Goulart etc., 2012) of $ 240,000.By reducing downstream cost, with non- The test of invasive biomarker supplements LDCT screening, can increase number of individuals (Peres, the J Natl of registration in LDCT screening Cancer Inst 105:1-2, 2013)。

Therefore, with using, any of above diagnosis or method of prognosis can not include that the screening system of miRNA expression analysis is tested The lung cancer of person is combined.Described system can be before miRNA expression analysis, use simultaneously or after.Currently known or send out later Existing any system benefits expected from the additionally analysis with MSC.The non-limiting examples of such system includes that blood is examined Test, x-ray, computerized tomography, positron emission tomography, thoracocentesis, BRO, thin Pin aspiration biopsy, thoracoscopy, thoracotomy or mediastinoscopy.In some embodiments, described system is Low dosage computerized tomography (LDCT).

E. Monitoring

Any of above method can be used for the risk monitoring the existence situation of lung cancer in experimenter and/or development lung cancer.When to not When the same time carries out any of above method available from the different biological sample of at least two of experimenter, monitor experimenter.One In a little embodiments, at least one of the different biological sample of at least two after lung cancer is treated by experimenter available from being subject to Examination person.The recurrence of such monitoring and evaluation lung cancer or risk of recurrence.

F. Treatment

Said method can be used for selecting treatment option.Terms used herein " treatment " means that description gives medicament to eliminate lung cancer Sign or symptom or reduce its seriousness.Alternatively or additionally, the illness that can occur at multiple positions, if at multiple positions At least one in administration therapy to illness, then obtain medical treatment.

According to an embodiment, said method can be used for selecting treatment option, if it is determined that feminine gender exists situation or wind If danger or determination low-risk in the combined method for predicting development or the risk with tumour, then experimenter is not to lung cancer Treat.In another embodiment, if it is determined that if during the positive exists situation or risk or determines in combined method Deng or excessive risk, then lung cancer is treated by experimenter.

Therefore, the method establishing lung cancer therapy option to experimenter is also provided.The method include using above-mentioned diagnosis and/ Or method of prognosis test experimenter and determine treatment option according to the result of described method.The method can further comprise administering to control Treat to experimenter in need.

G. MiRNA

24 miRNA can form existence situation (PAD) mark of existence situation (PD) label of lung neoplasm, aggressive lung neoplasm altogether Sign, risk (RD) label of lung neoplasm occurs and risk (RAD) label of aggressive lung neoplasm occurs.Table 1 describes at each mark This 24 miRNA that can exist in label.

Table 1

miRNA
	hsa-miR-16
hsa-miR-17
	hsa-miR-21
hsa-miR-101
	hsa-miR-126
hsa-miR-145
	hsa-miR-197
hsa-miR-221
	hsa-miR-320
hsa-miR-451
	hsa-miR-660
hsa-miR-106a
	hsa-miR-133a
hsa-miR-140-3p
	hsa-miR-140-5p
hsa-miR-142-3p
	hsa-miR-148a
hsa-miR-15b
	hsa-miR-19b
hsa-miR-28-3p
	hsa-miR-30b
hsa-miR-30c
	hsa-miR-486-5p
hsa-miR-92a

Table 2 provides the general introduction of the miRNA for all aspects of the invention.

Table 2

Following example are provided claimed invention is better described and are not construed as limiting the scope of the present invention.? When mentioning concrete material, purpose by way of example only, and it is not intended to limit the present invention.Those skilled in the art can develop equivalent Instrument or reagent, without the creative ability of utilization, and without departing from the scope of the present invention.

Embodiment

Following example relate to the microRNA labeling in the screening of computerized tomography lung cancer based on blood plasma The clinical function of symbol, also known as related MILD experimental study.There is provided the result of self-validation research, described checking research exists Random multicenter Italy lung detection (MILD) clinical testing of LDCT paired observation is collected expected Self smoke absorption experimenter In sample retrospective assessment 939 experimenters in determine preassignment miRNA labeling symbol (MSC) algorithm diagnosis performance (Pastorino etc., 2012).We demonstrate that, MSC has significantly diagnosis and prognosis performance.

Embodiment 1: method

A. colony is studied.Multicenter Italy's lung detection (MILD) test is a random prospective clinical trial, 2005 Year initiating, and state-run institute of oncology (Istituto Nazionale dei Tumori) raises 4 in Milan, 099 at present or The smoker of the past, at least 50 years old and in the first five years without cancer history: 2,376 (58%) is randomized to LDCT group (1190 Annually, 1186 once every two years LDCT) and 1,723 (42%) to observation group (Pastorino etc., 2012).In test All volunteer recruitings when (baseline) and each annual or when once every two years recalling, according to state of state-run institute of oncology, Milan Border examine and Ethics Committee (Internal Review and the Ethics Boards), by described (Boeri etc., 2011) whole blood is collected.

For this research, test raise without lung cancer individuality in from June, 2009 in July, 2010 collect 1,000 Part continuous print plasma sample is for determining that MSC's is specific.First measure plasma sample haemolysis (seeing below) with remove from The sample of the patient that may be polluted by haemocyte miRNA (Kirschner etc., 2011;Pritchard etc., 2012).At this In 1000 parts of samples, can not evaluate because of haemolysis for 130 parts.In remaining 870 experimenters, 594 (68%) belong to LDCT Group and 276 (32%) belong to observation group.For obtaining the group of the selectivity properties for determining MSC, examine Zi in September, 2012 The almost all patient for lung cancer of breaking obtains plasma sample (N=85).We are replacing the case of coupling and the big of comparison at preference In unselected experimenter series (it has greatly reduced comparison number and Research Ability), measurement negative predictive value (NPV).Right In this 85 patients 69, collect at least 1 part and can evaluate sample.For all patients, we consider closest drawing The sample that the LDCT of cancer diagnosis checks.Specifically, sample during diagnosis can be obtained to 50 patients, and 19 patients can be obtained Before get-disease sample (Fig. 1).Before-disease sample collection in 8-35 month before lung cancer detection, the median with 18 months is delayed Time.

B. MicroRNA profile analysis.From 200 μ l plasma sample mirVana PARISKit (Life Technologies, Ambion) extract total serum IgE, and elute in 50 μ l buffer solutions.MiRNA is expressed, elutes at 3 μ l RNA in, use the customization at 24 miRNA comprising duplicate point sample for the Multiplex Pools Protocol miniflow Card (Life Technologies, Applied Biosystems) is upper to be measured.For each sample, the Ct of individual miRNA uses ViiA7 software (Life Technologies, Applied Biosystems) is with threshold value 0.15 and automatic baseline determination.For defeated Entering to MSC, the mean value of the duplicate reading of the miRNA ratio of pre-determining is calculated by as described before (Boeri etc., 2011).

C. statistical analysis.The blind MSC risk score of the clinical effectiveness of individual subjects is committed in independent research The heart, Mario Negri research institute (Istituto Mario Negri) in Milan, and complete by the statistical analysis plan of preassignment Data analysis.The sensitiveness (SE) of MSC, specific (SP), positive predictive value (PPV) and negative predictive value (NPV) use computer Process, to evaluate the recognition performance of MSC, to LDCT that is whole and that study and observation group, will there is lung cancer after diagnosing Patient contrast and classify without disease subject.For diagnosis performance, by the individuality that is sorted in MSC-low-risk group with MSC-is medium or those in MSC-excessive risk group compare.In view of single positive and double positive tests, we also use at computer Manage SE and SP to be applied in combination binary MSC and LDCT.

In order to explain the prediction thing that the time dependence of MSC develops as disease, use Heagerty etc., Biometrics 56:337-344 (2000) and Zheng and Heagerty, Biometrics 63:332-341 (2007) The method describing, to from blood sample collect to different time interval (the 6th, the 12nd, 18 and 24 months) of pulmonary cancer diagnosis have rated SE, SP, PPV and NPV.

For determining the prognosis performance of MSC, check all three risk group, and in all 939 experimenters, according to MSC Obtain the Kaplan-Meier estimate as the date collected from blood sample for the Survival curves.We use Cox ratio accident The model evaluation survival inconsistency of MSC, it is also contemplated that the model that age and sex are adjusted further, and use computer disposal χ 21 between height/the medium and low MSC checks.

D. evaluating affects the haemolysis of plasma sample.The plasma sample that there is haemolysis removes from analysis subsequently, because blood Cell such as red blood cell (RBC) or hematoblastic haemolysis release pollute miRNA (Kirschner etc., 2011; Pritchard Deng 2012).Quality control twice (QC) is used to measure for this evaluation.First, the preanalysis step before RNA extracts In, complete spectrophotometry analysis by measurement in the absorbance (414nm, 541nm, 576nm) of different wave length, to identify In sample free hemoglobin existence situation and amount (Kirschner etc., 2011).

To all samples, by the expression of the miRNA that the haemolysis comprising in analysis miRNA labeling symbol is related to (MSC;Mir-451,486-5p, 16,92a), carry out second QC step to obtain even more big haemolysis sensitiveness.Molten The expression of the related miRNA of blood from the population mean of all samples more than the plasma sample of 2 standard deviations from subsequently Analysis is got rid of.There is the measurable sample through the haemolysis of spectrophotometry de termination measurement also arranged in the 2nd QC step Remove.Cancer versus's control sample is not observed and analyzes measurement through spectrophotometry de termination or by the related miRNA of haemolysis Haemolysis frequency or amount on difference.

In addition to the possibility of the pollution miRNA of haemolysis release, in blood plasma, the miRNA of differential expression can reflect simply Different blood counts (Pritchard, 2012).In order to analyze this it is assumed that by expressed by neutrophil leucocyte and RBC table The miRNA that reaches (Id.) full blood count (CBC) of the miRNA ratio that forms and 23 patients with lung cancer by studying from this obtains Neutrophil leucocyte and RBC level between ratio compare.As table 3 is reported, none quilt of miRNA ratio present in label Discovery has the significant correlation with respective CBC ratio.

The correlation of blood plasma miRNA ratio and blood count in 3. 23 patients with lung cancer of table.Neutrality in 23 samples The Pearson correlation coefficient of granulocyte contrast RBC miRNA ratio and neutrophil leucocyte contrast RBC counting is shown in table.Report Accuse statistically significant correlation (double tail p-values < 0.05).

Including the 2nd QC step of the analysis of the related miRNA of haemolysis can utilize haemolysis miRNA label additionally or in the alternative, It includes the miRNA that MSC as herein described is contained within.

i. Use miRNA label detection haemolysis: plasma collection

Collect whole blood sample, before adding EDTA, and process, be less than 1-2 hour at room temperature storage.Should avoid reducing temperature Lower storage, because it may result in non-specific release miRNA.Sample centrifuges 10 minutes with about 1250 x g at 4 DEG C, to separate Blood plasma, is carefully shifted, and avoids the material closest to lymphocyte ring simultaneously.By blood plasma again under the same conditions centrifuge and Be then divided into equal portions, must not deposited material ,-80 DEG C storage be up to 1 year or more long.

ii.There is not the MiRNA expression in the sample of haemolysis in the sample contrast that haemolysis occurs

In order to be developed for detecting the miRNA label of haemolysis, by 24 parts of plasma sample haemolysis.Haemolysis is evaluated by visual inspection. For the expression of one group of miRNA, use the overview of the sample of the miniflow card analysis generation haemolysis of customization.The sample of haemolysis will be there is In the expressing and expression ratio in 98 (view-based access control model assessment) samples that haemolysis does not occurs without diseased individuals of miRNA Relatively.

Four miRNA (miR-16, miR-451, miR-486-5p and miR-92a) include the exploitation at miRNA label In.Original Ct value (tolerance of expression) shows in table 4.Compared with the sample that haemolysis does not occurs, at the sample that haemolysis occurs In product, in addition to miR-140-3p, 4 miRNA, miR-16, miR-451, miR-486-5p and miR-92a, also significantly Increment regulates (p < 0.00l).

There is not the original Ct value of miRNA in the sample of haemolysis in the sample contrast that haemolysis occurs in table 4..

iii. MiRNA ratio is produced in haemolysis label

In order to use the difference of miRNA ratio haemolysis to occur and the sample that haemolysis does not occurs, analyze and comprise the molten in vitro of serial dilution The miRNA expression of the normal plasma samples of blood plasma sample.Calculate the four of each miRNA and the increment regulation of display in table 4 The ratio of each of individual miRNA (miR-16, miR-451, miR-486-5p and miR-92a).MiR-126、miR-15b、 MiR-221 and miR-30b is accredited as the miRNA with the ratio of four increment regulation miRNA with optimal haemolysis correlation (table 5).

Table 5. represents the blood plasma miRNA expression ratio of hemolysis in vitro.

Each value is expressed as log₂ (ratio)

Following formula is used to calculate average baselining miRNA ratio: from 98 without the plasma sample that haemolysis does not occurs of diseased individuals Average ratio+1.5 s.d. (standard deviation).These base line ratio are set as developing 16 in haemolysis miRNA label The cutoff of each of miRNA ratio.

iv. Use haemolysis miRNA label detection haemolysis

Haemolysis miRNA label can be used for detecting the haemolysis in plasma sample.Table 6 show by 8 miRNA (miR-126,15b, 30b, the 221st, the 451st, the 16th, 486-5p and 92a) list of 16 ratios that constitutes.Specifically, 4 miRNA, miR-451, MiR-486-5p, miR-16 and miR-92a, all overexpression in the sample that haemolysis occurs.This overexpression can be used as blood The indicant of haemolysis in slurry samples.If additionally, in the ratio of 16 shown in table 68 or more exceed cutoff, then blood plasma Sample is that haemolysis is positive.

Table 6. haemolysis miRNA label

v. Relatively haemolysis miRNA label and hemoglobin

For detecting the absorbance method of haemolysis

Hemoglobin known in the art has the absorbance wavelength of 414 nm.For science and clinical practice to analyze haemolysis A standard method be that spectral measurement at wavelength 414 nm for the free hemoglobin in plasma sample (uses absorbance threshold Value 0.2).In this experiment, absorbance normalization at 375 nm for the absorbance at 414 nm, to overcome at some samples In the high background signal of (for example in piarhemia sample).Ratio to the extinction at the absorbance at 414 nm and 375 nm, if Determine cutoff 1.4.

As shown in last column of table 5, the sample more than 1.40 for the A414nm/A375nm value is corresponding to wherein 16 miRNA In ratio at least 8 exceed the miRNA label of its respective cutoff.These set and are used for evaluating miRNA label in differentiation Ability in 24 plasma samples that haemolysis occurs and 98 (view-based access control model inspection) plasma samples that haemolysis does not occurs.miRNA Stamp methods shows the sensitiveness of 0.88 and 0.93 specific.With regard to sensitiveness, it is evident that visual assessment occurs haemolysis 24 samples there occurs haemolysis really.

Use and 60 plasma samples (red, orange or buff) of haemolysis are visually occurred and according to vision by select Check the independent series not occurring 43 plasma samples of haemolysis to constitute, compare haemolysis miRNA label and spectrophotometry side Method.With regard to sensitiveness (table 7), by two kinds of methods, it is positive that the sample of 5 haemolysis (red) is all identified as haemolysis.So And, when evaluating orange and buff sample, miRNA stamp methods is more sensitive (> 90% contrast spectrophotometry method 76%).

Table 7. compares spectrophotometry and miRNA result, to evaluate in 60 plasma samples that haemolysis visually occurs Haemolysis.

() is detected as occurring the overall percentage of haemolysis by each method

On the other hand, in 43 plasma samples that haemolysis does not occurs according to visual inspection (table 8), result display uses following two During the method for kind, 3 (7%) individual samples are that haemolysis is positive: in only spectrophotometry method, 1 (2%) individual sample is positive； With in only miRNA stamp methods, 2 (5%) individual samples are positive.Between two kinds of methods, 40 (93%) individual samples have molten Blood system class uniformity.In addition to being 3 cases of the positive in two kinds of methods, it is special that spectrophotometry method has 97.5% It is specific that the opposite sex and miRNA stamp methods have 95%.

Table 8. compares spectrophotometry and miRNA result, to evaluate 43, haemolysis (according to visual inspection) does not occurs Haemolysis in plasma sample.

() overall percentage；κ statistical value=0.63

E. MiRNA labeling symbol (MSC) algorithm.Describe MSC algorithm herein in detail.For developing this algorithm, in as described before INT/IEO lung cancer screening test (Boeri etc., 2011) before perspective collect or examining from INT/IEO lung cancer screening test In the training set of the sample of patients with lung cancer when disconnected, the 4,950 of 100 different miRNA that in comfortable blood plasma, (stablizing) is circulated Individual ratio starts, and the different genes producing 24 different miRNA expresses ratio.MSC algorithm for this preassignment studied Exploitation is from the refine in two ways of research before.

First, the sample that can detect haemolysis is removed (Boeri etc., 2011) in original training set.This publication it After, researcher reports the measurement of expression accurately that haemolysis affects miRNA in blood plasma and blood serum sample.Therefore, it is used for for generation The optimal training set of MSC, owing to can detect the hemoglobin miRNA level related with haemolysis by spectrophotometry analysis Increasing (as described herein), 5 in original training set sample is excluded.MiRNA ratio is subsequently used for optimum training collection to open Sending out disease risks (RD), there is situation (PD), affecting conditions risk (RAD) and affecting conditions and there is situation (PAD in disease； Table 9) miRNA label.

The miRNA-ratio label of table 9. refine and corresponding cutoff (log2).

RD: disease risks；RAD: affecting conditions risk；PD: disease exists situation；PAD: affecting conditions exists feelings Condition.

Set up ratio cutoff at-80 DEG C of storages plasma sample of at least 1 year and at most 5 years.

Second method from the MSC algorithm development being different from this research studied before is, uses from MILD test It is not included in 84 plasma samples without the training set of diseased individuals in 939 experimenter's checking groups, produce the ratio of pre-determining Rate cutoff obtaining >=80% specific.

The plasma sample from single experimenter is used to replace storehouse (at Boeri etc., use in 2011), it is allowed to Wo Menkao Consider all 24 miRNA initially identifying in training set and produce cutoff accurately.Therefore, this research can include mir- 101st, 145 and 133a, it is excluded at it because of high variability between the experimenter in the comparison storehouse for this research Front checking is concentrated.

In order to set up three horizontal classification of risks (MSC is low, medium and high) to disease, training set is also used for establishment and exceedes and recognized It for being the minimum ratio number of respective cutoff that is positive and that need: be 10/27 for RD, is 9/27 for PD, for RAD is 14/28, it is 14/28 for PAD.Then three horizontal MSC press defined below: if RDneg is ∩ PDneg ∩ RADneg ∩ PADneg, then be low-risk (L)；It if RDpos is U PDpos ∩ RADneg ∩ PADneg, then is medium risk (I)；Or It if RADpos is U PADpos, then is excessive risk (H).The risk group of these preassignment is subsequently used for testing from MILD sieve Diagnosis in the independent sets of 939 experimenters of choosing test and prognosis performance.

F. the feature of experimenter.939 experimenters' with appreciable plasma sample raising in MILD test Feature (2005-2012; LDCT: N=652;Observe: N=287), including 69 patients with lung cancer and 870 tested without lung cancer Person, points out (table 10) according to age, sex and smoking state, duration and cigarette number/sky.Patients with lung cancer age is more than nothing Patients with lung cancer (p < 0.0001) and higher (81.2% vs. 63.3% of masculinity proportion; p=0.0029).Smoking state is having cancer Or without between cancer group without significant difference, but time that the experimenter of developing cancer smoking is longer (p < 0.0001).

Table 10: according to age, sex and smoking, 69 lung cancer experimenters and 870 distributions without lung cancer experimenter.In how Heart Italy's lung detection (MILD) research, 2005-2012.

For cancer patient, from random, the median time to diagnosis is 29 months (scopes 1-82) and sample extremely from blood plasma The median time of diagnosis is 2 months (scope 0-35).Without in lung cancer experimenter, when at random to the median of blood plasma sampling Between be 44 months (scopes 0-58) and from blood plasma sampling to the median time finally followed up a case by regular visits to be 27 months (scope 3-41).

The diagnosis of embodiment 2:MSC and prognosis performance

For before the diagnosis or the valuable blood plasma that obtains from 939 experimenters crossing over LDCT and observation group when diagnosis Sample, uses the MSC algorithm based on low, the medium and high risk preassignment with cancer group of the determination method of real-time RT-PCR to carry out Analyze.To all 939 experimenters, check MSC risk group according to lung cancer generation, lung cancer death and tumor stage (table 11).MSC Medium and high 60 correctly having classified in 69 patients with lung cancer, wherein 87% SE, 81% SP, 27% PPV and 99% NPV. In 19 patients with lung cancer following up a case by regular visits to period death, 18 is the MSC test positive, wherein 95% SE, 81% SP, 10% PPV With 100% NPV.Follow up a case by regular visits to period do not observe due to and the death of the reason that non-lung cancer.To lung cancer detection ratio in two groups MSC diagnosis performance relatively is similar, is wherein respectively 88% SE, 80% SP, 31% PPV, 99% NPV to LDCT and observation group With 82% SE, 83% SP, 16% PPV, 99% NPV.

Table 11. accords with (MSC) according to lung cancer illness rate, lung cancer death and lung cancer stage and miRNA labeling, 69 lungs Cancer experimenter and 870 distributions without lung cancer experimenter, and corresponding sensitiveness (SE), specific (SP), positive predictive value And negative predictive value (NPV) (PPV).Multicenter Italy's lung detection (MILD) research, 2005-2012.

In all three MSC risk group, it was observed that disease causes the visible trend of ratio of death, wherein respectively with The ratio of the lung cancer death related with height low, medium increases (p=0.0336).MSC risk group and each tumor stage (I, II- III or IV；Table 11) not significant correlation (p=0.40).

(χ between MSC risk group and histological subtypes²=1.60, p=0.4485) and at gland cancer and squamous cell carcinoma Between (χ²=0.55, p=0.759) do not observe significant difference.The time dependence analysis of the diagnosis performance of MSC is shown in Between blood sampling and pulmonary cancer diagnosis the 6th, the 12nd, 18 with SE, SP, PPV value (table 12) similar with NPV at 24 months intervals.

Table 12. miRNA labeling symbol (MSC) time dependence analysis, and from blood sampling to lung cancer detection the 6th, 12nd, 18 and the sensitiveness (SE), specific (SP), positive predictive value (PPV) and the negative predictive value (NPV) that within 24 months, calculate.

The supplemental diagnostics performance of embodiment 3:LDCT and MSC

Limiting analysis is to 652 experimenters altogether in LDCT group, and LDCT identifies 46 in 58 lung cancer experimenters, lacks Do not detect 3 patients in 251 experimenters of lung brief summary and because 9 patients (table 13) of the interval cancer disease of the SE of 79%. The three case cancers of " apneumia brief summary " include that a case non-physical focus, a case indulge diaphragm adenopathy and a case pleural effusion.The MSC of preassignment Binary risk group (considering that high and medium contrast is low) identifies 40 cases in the cancer of 46 case LDCT-detections, in 9 case interval cancer diseases 8 cases and whole 3 " apneumia brief summary " experimenters.

LDCT has to non-calcified brief summary > the invalid subgroup of the clinic of 5 mm 81% SP and related false positive rate 19.4% (115/594) (table 13).When considering double positive (LDCT and MSC) experimenter, false positive rate is reduced to 3.7% (22/594), wherein SE reduces (40/58,69%).On the contrary, consider that there is at least one positive test (LDCT or MSC) Experimenter is for, when positive, being applied in combination LDCT and MSC and identifying 57 in 58 cases, and wherein SE is 98% and SP is 65%. On the other hand, MSC detection 9 cases in 11 (82%) example lung cancer (table 13) present in observation group.

Embodiment 4:MSC risk group associates with survival rate

From to having the plasma sample that all experimenters (N=939) following up a case by regular visits to collect 3 years, complete to analyze with determine three pre-really The prognosis performance of fixed MSC risk group, with prediction of overall survival rate.To the experimenter with low MSC, two annual survival rates are 100%, it is 98% to medium MSC and is 87% to high MSC, and to low, medium and high, three annual survival rates are the 100%th, 97% and respectively 77% (Fig. 2).Between height/medium and low MSC, the difference of survival rate is statistically significant (χ² =49.53, P< 0.0001).After adjusting age and sex, inconsistency is still significant (χ²=12.57, P=0.0004)。

Embodiment 5: analyze

The diagnostic characteristic of the hypersensitivity related to the NPV of 99% shows, MSC is the screening test through verifying clinically.Additionally, MSC is confirmed by time dependence analysis as the diagnosis performance of the prediction thing that lung cancer develops.

MSC identifies the experimenter with the high likelihood that disease causes death.As binary diagnostic, for leap two 939 experimenters of group, disease is caused death to have the SE of the 95% and NPV of 100% by MSC.Additionally, MSC risk group with for The 3 dramatically different annual survival rates of whole group of 939 experimenters are related (is respectively the 100%th, 97% for low, medium and high MSC With 77%).These discoveries confirm that blood plasma miRNA identifies the pernicious of tumour and aggressive.

Three little European random experiments, including MILD, the death rate that it has been reported non-significant so far reduces (Infante Deng 2009;Saghir etc., 2012;Pastorino etc., 2012).Country's lung screening test (NSLT), raises 53 for one, 454 people take turns, with the 3 of contrast chest radiogram, the random screening that LDCT screens annually and test it was confirmed lung cancer is dead Rate of dying reduces by 20% (Aberle etc., 2011).After taking turns screening 3, the experimenter of 24.2% is classified as the positive, and wherein 96.4% These experimenters be false positive, need to screen 320 experimenters to prevent 1 case lung cancer death.

In the system review of all of randomized clinical trial of the benefit and harm that check LDCT screening, average brief summary is examined Going out rate is 20%, and the wherein brief summary of 90% is optimum (Bach etc., JAMA 307:2418-2429,2012).In this study, It 74% (485) is low-risk that MSC has classified in 652 individualities of LDCT group: 478 entitled true negatives and 7 entitled false negatives. MSC can identify by LDCT fail detection 9 case interval cancer diseases in 8 cases.Therefore, integration (at least one example of MSC and LDCT Test the positive) by raisings screening sensitiveness, from the 87% and independent LDCT of independent MSC 84% to 98% (57/58), false positive rate It is 35%.On the contrary, in view of double positive subjects (MSC and LDCT is positive), the integration of MSC and LDCT will reduce LDCT screening False positive rate more than 5-times.Particularly, compared with the 19.7% (115/594) of independent LDCT, double false positives MSC and LDCT's Frequency is only 3.7% (22/594), but reduces SE to 69%.

Therefore, MSC can be supplemented LDCT screening by reducing false positive results, and diagnosis algorithm can be made to mark more Standardization, thus reduce health care cost.Additionally, in view of lacking 3 annual death rates for low MSC experimenter, can propose to having The individuality of low MSC repeats MSC rather than LDCT further.This blind research of the determination method based on blood plasma of preassignment represents LDCT screening test tests the maximum research of biomarker, and confirms the outstanding diagnosis performance of LDCT when combining with MSC.

Claims

1. determining the method for the existence situation of lung neoplasm in experimenter, described method includes:

D other miRNA to repeating step (a) to (c), is allocated to expressing ratio by () until (1) at least 9 miRNA Just mark, or (2) after comparing the expression ratio of 27 miRNA pair, be just assigned to expressing ratio less than 9 miRNA Scoring,

2. the process of claim 1 wherein described miRNA to include 106a/140-5p, 106a/142-3p, 126/140-5p, 126/142-3p、133a/142-3p、140-5p/17、142-3p/148a、142-3p/15b、142-3p/17、142-3p/21、 142-3p/221,142-3p/30b and 320/660, or its inverse ratio.

3. the process of claim 1 wherein described miRNA to farther including 106a/660,106a/92a, the 126/660th, 140- 5p/197、140-5p/28-3p、142-3p/145、142-3p/197、142-3p/28-3p、17/660、17/92a、197/660、 197/92a, 19b/660 or 28-3p/660, or its inverse ratio.

4. the method for claim 2, wherein said miRNA is to farther including 106a/660,106a/92a, the 126/660th, 140- 5p/197、140-5p/28-3p、142-3p/145、142-3p/197、142-3p/28-3p、17/660、17/92a、197/660、 197/92a, 19b/660 and 28-3p/660, or its inverse ratio.

5. determine the method for the existence situation of aggressive lung neoplasm in experimenter, comprising:

D other miRNA to repeating step (a) to (c), is allocated to expressing ratio by () until (1) at least 14 miRNA Just mark, or (2) after comparing the expression ratio of 28 miRNA pair, be assigned to expressing ratio less than 14 miRNA Just mark,

6. the method for claim 5, wherein said miRNA is to including 106a/16, the 106/660th, the 16/17th, the 16/320th, the 17/660th, 197/30b, 197/30c, the 320/451st, 320/486-5p and 320/660, or its inverse ratio.

7. the method for claim 5, wherein said miRNA is to farther including 106a/451,106a/486-5p, the 126/451st, 126/486-5p、126/660、140-5p/197、16/197、17/451、17/486-5p、197/451、197/486-5p、197/ 660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p, or its inverse ratio.

8. the method for claim 6, wherein said miRNA is to farther including 106a/451,106a/486-5p, the 126/451st, 126/486-5p、126/660、140-5p/197、16/197、17/451、17/486-5p、197/451、197/486-5p、197/ 660th, 197/92a, 19b/451,19b/486-5p, 19b/660,28-3p/451 or 28-3p/486-5p, or its inverse ratio.

9. determining in experimenter the method for risk lung neoplasm occur, described method includes:

D other miRNA to repeating step (a) to (c), is allocated to expressing ratio by () until (1) at least 10 miRNA Just mark, or (2) after comparing the expression ratio of 27 miRNA pair, be assigned to expressing ratio less than 10 miRNA Just mark,

10. the method for claim 9, wherein said miRNA to include 133a/92a, 15b/21,15b/30b, 15b/30c, 16/ 197 and 28-3p/451, or its inverse ratio.

The method of 11. claims 9, wherein said miRNA to farther include 101/140-3p, 106a/451,106a/660, 106a/92a、126/660、133a/451、133a/660、140-3p/660、142-3p/15b、15b/451、15b/660、17/ 451st, the 17/660th, 17/92a, 197/19b, the 197/451st, the 197/660th, 197/92a, 19b/660,28-3p/660 or 320/660, Or its inverse ratio.

The method of 12. claims 10, wherein said miRNA is to farther including 101/140-3p, 106a/451,106a/ 660、106a/92a、126/660、133a/451、133a/660、140-3p/660、142-3p/15b、15b/451、15b/660、 17/451st, the 17/660th, 17/92a, 197/19b, the 197/451st, the 197/660th, 197/92a, 19b/660,28-3p/660 and 320/ 660, or its inverse ratio.

Occurring the method for the risk of aggressive lung neoplasm in 13. determination experimenters, described method includes:

The method of 14. claims 13, wherein said miRNA is to including 106a/142-3p, 126/142-3p, the 126/21st, 126/ 92a, 142-3p/17,142-3p/197,142-3p/28-3p, 197/19b, 197/660 and 28-3p/660, or its inverse ratio.

The method of 15. claims 13, wherein said miRNA to farther include 106a/451, the 126/451st, the 145/197th, 17/ 451、197/21、197/30b、197/30c、197/451、197/92a、19b/451、21/221、21/28-3p、28-3p/30b、 28-3p/30c, 28-3p/451,28-3p/92a, 320/451 or 320/92a, or its inverse ratio.

The method of 16. claims 14, wherein said miRNA to farther include 106a/451, the 126/451st, the 145/197th, 17/ 451、197/21、197/30b、197/30c、197/451、197/92a、19b/451、21/221、21/28-3p、28-3p/30b、 28-3p/30c, 28-3p/451,28-3p/92a, 320/451 and 320/92a, or its inverse ratio.

17. methods predicting the risk developing or having lung neoplasm in experimenters, comprising:

A () determines the existence situation label of the lung neoplasm of claim 1；

B () determines the existence situation label of the aggressive lung neoplasm of claim 5；

C () determines the risk label of the appearance lung neoplasm of claim 9；

D () determines the risk label of the appearance aggressive lung neoplasm of claim 13；

If g the existence situation label of the aggressive lung neoplasm of () at least step (b) is the positive, or the appearance invasion and attack of step (d) Property lung neoplasm risk label be the positive, then experimenter is categorized as development or there is the excessive risk of lung neoplasm.

18. claims the 1st, the 5th, the method any one of 9 or 13, wherein said cutoff is determined as from multiple control samples Average ratio.

19. claims the 1st, the 5th, the method any one of 9 or 13, wherein said cutoff is determined as from multiple control samples One standard deviation of average ratio +/-.

20. claims the 1st, the 5th, the method any one of 9 or 13, wherein said cutoff is determined as from multiple control samples Median ratio.

21. claims the 1st, the 5th, the method any one of 9 or 13, wherein miRNA is to expressing ratio by using reverse transcriptase-poly- The cDNA copy that synthase chain reaction (RT-PCR) prepares each miRNA of each miRNA centering measures.

The method of 22. claims 21, wherein expresses ratio and uses real-time PCR to measure further.

The method of 23. claims 22, wherein expresses ratio and uses TaqMan probe to measure further.

24. claims the 1st, the 5th, the method any one of 9 or 13, wherein said biological sample is biofluid.

The method of 25. claims 24, wherein said biofluid is urine, blood, blood plasma or serum.

The method of 26. claims 24, wherein said biofluid is blood plasma or serum.

The method of 27. claims 26, wherein measures haemolysis in biological sample, and if there is the haemolysis that can measure, then institute State sample and do not carry out described method.

The method of 28. claims 27, wherein through spectrophotometry measurement haemolysis.

The method of 29. claims 27, wherein by analyzing what the haemolysis of increment regulation in the sample that haemolysis occurs was related to The expression of miRNA measures haemolysis.

The method of 30. claims 29, the related miRNA of wherein said haemolysis include miRNA miR-451, miR-486-5p, Multiple in miR-16, miR-92a or miR-140-3p.

The method of 31. claims 29, the related miRNA of wherein said haemolysis be miR-451, miR-486-5p, miR-16 and miR-92a。

The method of 32. claims 29, farther includes to measure multiple corrections of non-increment regulation in the sample that haemolysis occurs The expression of miRNA, with the expression normalization of the miRNA that haemolysis is related.

The method of 33. claims 32, wherein said correction miRNA includes miR-126, miR-15b, miR-221 and miR- 30b。

The method of 34. claims 33, wherein haemolysis is measured by following further:

A () determines by each of miR-451, miR-486-5p, miR-16 and miR-92a in sample with miR-126, miR- The expression ratio of each of be composed of 16 miRNA pair of each of 15b, miR-221 and miR-30b；

B () compares each of 16 expression ratios from step (a) and expresses ratio from multiple control samples to each Multiple corresponding miRNA pair average ratio measure cutoff；

C () is for each of 16 miRNA pair, if the expression ratio of step (a) exceedes the cutoff of step (b), then right The distribution of described ratio is just marked；If or the expression ratio of step (a) is not less than the cutoff of step (b), then to described ratio Distribution anon-normal scoring；With

D described sample is classified by () by following: if in (i) 16 ratios 8 or more exceed cutoff, then have Haemolysis；Or (ii) is if exceeding cutoff less than 8 in 16 ratios, then do not have haemolysis.

The method of 35. claims 29, wherein haemolysis is also measured through spectrophotometry de termination.

36. claims the 1st, the 5th, the method any one of 9 or 13, farther include to use do not include miRNA expression analysis be System, before miRNA expression analysis or simultaneously, screens experimenter for lung cancer.

The method of 37. claims 36, wherein said system includes blood test, x-ray, computerized tomography, positive electricity Sub-emission tomography, thoracocentesis, BRO, fine needle aspiration biopsy, thoracoscopy, thoracotomy Art, mediastinoscopy.

The method of 38. claims 37, wherein said system is low dosage computerized tomography (LDCT).

39. claims the 1st, the 5th, the method any one of 9 or 13, the life different at least two of experimenter of wherein said method Thing sample is carried out.

The method of 40. claims 39, at least one of the different sample of wherein said at least two experimenter to lung cancer Obtain from experimenter after treating.

41. claims the 1st, the 5th, the 9th, the method any one of 13 or 17, wherein if it is determined that the existence situation of feminine gender or risk, or If it is determined that low-risk, then lung cancer is not treated by experimenter.

42. claims the 1st, the 5th, the 9th, the method any one of 13 or 17, wherein if it is determined that the existence situation of the positive or risk, or If it is determined that medium or excessive risk, then lung cancer is treated by experimenter.

The method that 43. couples of experimenters establish lung cancer therapy option, described method includes using claim the 1st, the 5th, the 9th, in 13 or 17 The method test experimenter of any one, and determine treatment option according to the result of described method.